VCP-Based Vectors for Algal Cell Transformation

ABSTRACT

Provided herein are exemplary vectors for transforming algal cells. In exemplary embodiments, the vector comprises a Violaxanthin-chlorophyll a binding protein (Vcp) promoter driving expression of an antibiotic resistance gene in an algal cell. Embodiments of the invention may be used to introduce a gene (or genes) into the alga  Nannochloropsis,  such that the gene(s) are expressed and functional. This unprecedented ability to transform  Nannochloropsis  with high efficiency makes possible new developments in phycology, aquaculture and biofuels applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present continuation application claims the benefit and priority ofU.S. Non-Provisional patent application Ser. No. 12/480,635 filed onJun. 8, 2009, titled “VCP-Based Vectors for Algal Cell Transformation,”which in turn claims the benefit and priority of U.S. Provisional PatentApplication Ser. No. 61/059,672 filed on Jun. 6, 2008, titled “VCP-BasedVector for Nannochloropsis Transformation,” all which are herebyincorporated by reference.

The present continuation application is related to U.S. Non-Provisionalpatent application Ser. No. 12/480,611 filed on Jun. 8, 2009, titled“Transformation of Algal Cells,” which is hereby incorporated byreference.

REFERENCE TO SEQUENCE LISTINGS

The present application is filed with sequence listing(s) attachedhereto and incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to molecular biology, and more specifically, tothe expression of exogenous DNA elements in algal cells.

2. Description of Related Art

Manipulating the DNA of a cell may confer upon the cell new abilities.In many cases, the genetic manipulation is carried out by introducingfunctional DNA that was prepared outside the cell using moleculartechniques. For example, a transformed cell (i.e., a cell that hastaken-up exogenous DNA) may be more robust than the wild-type cell. Formany so-called model biological systems (i.e., well-studied organisms),the DNA elements for transformation have been developed. For otherorganisms, of which less is known, transformation is a major milestonethat must be achieved to facilitate genetic engineering. Many algalspecies fall into the category of non-model organisms, with recalcitrantcell walls that make them notoriously difficult to transform.Accordingly, there is a need for an expression vectors forNannochloropsis transformation.

SUMMARY OF THE INVENTION

Provided herein are exemplary vectors for transforming algal cells. Inexemplary embodiments, the vector comprises a Violaxanthin-chlorophyll abinding protein (Vcp) promoter driving expression of an antibioticresistance gene in an algal cell. Embodiments of the invention may beused to introduce a gene (or genes) into the alga Nannochloropsis, suchthat the gene(s) are expressed and functional. This unprecedentedability to transform Nannochloropsis with high efficiency makes possiblenew developments in phycology, aquaculture and biofuels applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a sequence of the genomic DNA of Nannochloropsis oceanica.

FIG. 1B shows an exemplary DNA transformation construct representing thefunctional insert of the PL90 vector.

FIG. 2 shows an exemplary nucleotide sequence (SEQ ID NO:1) for theinsert of the PL90 vector.

FIG. 3 shows an exemplary nucleotide sequence (SEQ ID NO:2) wherein theSh ble gene of the PL90 vector was replaced with a gene conferringresistance against hygromycin B.

FIG. 4 shows an exemplary nucleotide sequence (SEQ ID NO:3) wherein theSh ble gene of the PL90 vector was replaced with a gene conferringresistance against blastocidin.

FIG. 5 shows the number of exemplary transformed algal cell coloniesobtained when cells from a single transformation experiment have beenplated under varying light conditions.

FIG. 6 shows the number of exemplary transformed algal mutants obtainedunder varying zeocine concentrations.

FIG. 7 shows the molecular analysis of exemplary transformed algal cellstransformed with the PL90 vector and grown in the presence of zeocine.

FIG. 8 shows an approximately 400 base pair fragment that indicates theexemplary linearized PL90 vector is stably integrated within the genomeof Nannochloropsis oceanica.

DETAILED DESCRIPTION OF THE INVENTION

Transformed algae may be useful in aquaculture production. Thetransformation of small algal cells with tough membranes, however, isdifficult to achieve. Embodiments of the present invention are useful inthe efficient transformation of Nannochloropsis, a microalga of about3-5 micrometers in size.

Various exemplary embodiments provided herein use aViolaxanthin-chlorophyll a binding protein (Vcp) promoter in atransformation construct to drive high levels of gene expression inalgal cells at low light intensities. The transformation construct maybe introduced within an algal cell or within an algal genome using oneof the exemplary methods described in U.S. Non-Provisional patentapplication Ser. No. 12/480,611 filed on Jun. 8, 2009, titled“Transformation of Algal Cells,” which is hereby incorporated byreference. An exemplary Nannochloropsis transformant in a logarithmicgrowth phase, plated onto F/2 media, and allowed to incubate at variouslight intensities for about two months, demonstrated that moretransformant colonies could grow at high levels (about 25 ug/ml) ofzeocine at low light levels. Thus, the Vcp promoter is active at lowerlight intensities, such that a transformation construct comprising a Vcppromoter may be useful in aquaculture ponds receiving less light, suchas in the case of algae grown deep in a pond. Additionally, a Vcppromoter may be useful in modulating the expression of genes governed bythe Vcp promoter, by varying the intensity of incident light.

FIG. 1A shows the sequence genomic DNA of Nannochloropsis oceanica,which includes the Vcp gene and regulatory elements. Please note thatfor illustration purposes, 2 exons and 1 intron within B3 areillustrated. In fact, the gene harbors more than these components. Thesequenced genomic DNA has the structure A-B-C, where B is the DNAencoding the Vcp gene (including introns), A is the DNA sequence infront of the Vcp gene, and C is the DNA sequence after the Vcp gene.

Sequence A includes the promoter which drives expression of the Vcpgene. The region from transcription start to translation start (thestart ATG triplet) is the 5′-untranslated region A3. The sequencepreceding the start methionine comprises A1, A2 and A3. The startmethionine B1 is immediately followed by an intron B2 and the remainingexons and introns B3 of the Vcp gene. The Vcp gene ends with the stopcodon B4. The sequence downstream of the Vcp gene (called C), includesthe untranslated region C1, a polyadenylation signal C2, the stop oftranscription C3 and downstream DNA sequence C4.

FIG. 1B shows an exemplary DNA transformation construct representing thefunctional insert of the PL90 vector. Here, part B3 (FIG. 1A) wasreplaced with the reading frame of the Sh ble gene found inStreptoalloteichus hindustanu, yielding the PL90 vector as describedherein.

FIG. 1B may also be used to show the structure of the various exemplaryvector constructs PL90, H8 and B9 as described herein. The differencebetween the three exemplary vector constructs is the type of selectionmarker gene (SG) used: the Sh ble gene (PL90), the hygromycin Bphosphotransferase gene (H8), or the blastocidin S deaminase (B9) gene.

EXAMPLE ONE

We identified a Vcp (violaxanthine chlorophyll a binding protein) genein a public nucleotide database (NCBI) for a Nannochloropsis strain(http://www.ncbi.nlm.nih.gov/nuccore/2734863). We constructed primersagainst this gene and recovered the genomic area in front of and behindthe gene. We designed a DNA transformation construct replacing part B3of the genome (FIG. 1A) with the reading frame of the Sh ble gene fromStreptoalloteichus hindustanus (which confers resistance against thedrug bleomycine), yielding the exemplary PL90 vector. This exemplaryconstruct is illustrated in FIG. 1B.

We retained the start methionine of the Sh ble gene. We introduced asecond start methionine immediately after the intron B2, thus thetranslation product includes spliced transcripts of the ble gene withtwo consecutive methionines. The spliced transcript thus starts with theamino acids “MIM,” with the second methionine being the beginning of theSh ble gene. This exemplary construct was linearized within the vectorby restriction digestion and used for the transformation ofNannochloropsis oceanica.

FIG. 2 shows an exemplary nucleotide sequence (SEQ ID NO:1) for theinsert of the PL90 vector. 202 represents A from FIGS. 1A-1B, which isthe DNA sequence in front of the Vcp gene. 204 represents the leftintron border of the first Vcp intron. 206 represents the startmethionine of the Vcp gene. 208 represents the beginning of theselection marker gene (i.e., the beginning of the Sh ble gene, ATG). 210represents an introduced artificial sequence, TT. 212 represents theright intron border of the first Vcp intron. 214 represents the stopcodon of the selection marker gene, TAA. 216 represents wherepolyadenylation occurs, after the CCGCCC sequence. 218 represents C fromFIGS. 1A-1B, which is the DNA sequence downstream of the Vcp gene.

FIG. 3 shows an exemplary nucleotide sequence (SEQ ID NO:2) wherein thesh ble gene of the PL90 vector was replaced with a gene conferringresistance against hygromycin B. 302 represents A from FIGS. 1A-1B,which is the DNA sequence in front of the Vcp gene. 304 represents theleft intron border of the first Vcp intron. 306 represents the startmethionine of the Vcp gene. 308 represents the beginning of theselection marker gene (i.e., the beginning of the hygromycin Bphosphotransferase gene, ATG). 310 represents an introduced artificialsequence, TT. 312 represents the right intron border of the first Vcpintron. 314 represents the stop codon of the selection marker gene, TAA.316 represents where polyadenylation occurs, after the CCGCCC sequence.318 represents C from FIGS. 1A-1B, which is the DNA sequence downstreamof the Vcp gene.

FIG. 4 shows an exemplary nucleotide sequence (SEQ ID NO:3) wherein theSh ble gene of the PL90 vector was replaced with a gene conferringresistance against blastocidin. 402 represents A from FIGS. 1A-1B, whichis the DNA sequence in front of the Vcp gene. 404 represents the leftintron border of the first Vcp intron. 406 represents the startmethionine of the Vcp gene. 408 represents the beginning of theselection marker gene (i.e., the beginning of the blasticidin-Sdeaminase gene, ATG). 410 represents an introduced artificial sequence,TT. 412 represents the right intron border of the first Vcp intron. 414represents the stop codon of the selection marker gene, TAA. 416represents where polyadenylation occurs, after the CCGCCC sequence. 418represents C from FIGS. 1A-1B, which is the DNA sequence downstream ofthe Vcp gene.

The exemplary vectors PL90 (FIG. 2), H8 (FIG. 3) and B9 (FIG. 4) areuseful for the transformation of Nannochloropsis. Selection occurred on2 μg/ml zeocine (for vector PL90), 300 μg/ml hygromycin B (vector H8),or 50 50 μg/ml blasticidin S (vector B9).

Resistant colonies were only obtained when the appropriate vectors wereused with the corresponding antibiotic. The success of transformationwas checked and proofed via PCR on genomic DNA isolated from potentialtransformed colonies obtained by transformation with the Vcp basedvectors described herein.

The Vcp promoter described herein drives expression of the Vcp ofNannochloropsis, a protein which is expressed in different levels atdifferent physiological conditions. Algal cells acclimated to higherlight intensities for example typically accumulate less light harvestingcomplexes than those acclimated to lower light intensities. We thuswanted to find out if the Vcp promoter described herein confersresistance to higher concentrations of zeocine (thus indicating higherexpression levels of the Vcp promoter-driven Sh ble gene) in differentlight intensities. We thus transformed Nannochloropsis cells with theconstruct shown in FIG. 2 and allowed selection on agar plates indifferent light intensities. For this purpose, we plated a singletransformation experiment on agar plates containing 25 μg/ml zeocine, or25 μg/ml zeocine but the cells plated within top-agarose, or on agarplates containing no zeocine at all. Wild type (no Sh ble gene) wasconsistently killed completely at 2 μg/ml zeocine. Resistance to higherconcentrations of zeocine (e.g. 25 μg/ml) indicates higher expressionlevel of the selection gene Sh ble at lower irradiance levels.

FIG. 5 shows the number of exemplary transformed algal cell coloniesobtained when cells from a single transformation experiment have beenplated under varying light conditions. FIG. 5 shows that the number ofcolonies (which is equal to the number of transformed cells which canstand concentrations of zeocine as high as 25 μg/ml) increases withdecreasing light intensities. The highest number of transformants wasobtained at low light intensities at 5 μE (μmol photons/(m2*s)). Theresult indicates that the exemplary construct utilized (as shown in FIG.2) has a higher level of gene expression at lower light intensities thanat higher light intensities. Accordingly, the exemplary constructs shownin FIGS. 2-4 might be utilized for the expression of genes modulated bythe intensity of light.

FIG. 6 shows the number of exemplary transformed algal mutants obtainedand showing fifty percent (50%) or more growth at a given zeocineconcentration but less than 50% at the next highest tested zeocineconcentration. FIG. 6 illustrates the frequency of 96 clones obtainedwith the transformation vector PL90 showing more than 50% growth (in aliquid assay monitoring growth via OD750) at a certain zeocineconcentration, but less than 50% at the next higher tested zeocineconcentration.

Please note, wild-type cells and control cells (those transformed withpJet1 NOT containing a construct) never form colonies on zeocineconcentrations 2 μg/ml or above, nor is there any detectable growth inliquid culture at such concentrations of zeocine. These resultsdemonstrate that the ble gene driven by the Vcp promoter in theconstruct confers resistance against zeocine concentrations up to 75μg/ml, while wild-type cells consistently cannot survive concentrationsabove 2 μg/ml.

We subsequently replaced the reading frame of the she ble gene withgenes conferring resistance to hygromycin B (transformation constructH8) and blastocidin (transformation construct B9) and used these vectorsfor transformation of Nannochloropsis oceanica. Again, we observed manytransformation events (while the control did not show any coloniesdeveloping). Selection conditions were identical as for the PL90transformation vector, with the exception that hygromycin B at 300 μg/mlor blastocidin S at 50 μg/ml were used.

FIG. 7 shows the molecular analysis of exemplary transformed algal cellstransformed with the PL90 vector and grown in the presence of zeocine.12 randomly picked colonies were derived from a transformation eventwith the vector PL90 and selection on zeocine (2 μg/ml). A controlcolony was obtained from a plate with wild-type colonies. Cells wereresuspended in buffer (1× yellow tango buffer from Fermentas) andincubated with DNAse in order to digest possible residual extra cellularPL90 DNA used for the transformation event. The cells were then washedtwice in seawater and resuspended in Millipore water and heated to 95 Cin order to bring the intracellular DNA into solution.

A standard PCR employing Sh ble gene primers (113 Ble for short ATG GCCAAG TTG ACC AGT GCC GT, 111 Ble rev short TTA GTC CTG CTC CTC GGC CACGAA) utilizing a taq polymerase was performed on lysates of the 12colonies obtained after transformation (colonies 1-12), of the controlwild type colony without (negative control NC) or with (positive controlPC) vector PL90 added.

The PCR reactions were separated on an ethidium bromide containing 1%agarose gel in TAE buffer. The 12 colonies from the transformation eventcontained the Sh ble gene (˜375Nt), as does the positive control, butNOT the negative control. We conclude that that the she ble gene iscontained within the cells and that the vector PL90 was used as atransformation construct to confer resistance against zeocine.

We then performed a Tail PCR (Liu and Hang 1998) employing the primersshown in the table below:

151 pJet1-prim CTTGCTGAAAAACTCGAGCCATCCG 152 pJet1-secAtggtgttcaggcacaagtgttaaagc 153 pJet1-tert- Ggtttagaacttactcacagcgtgatgc

The primers shown above correspond to the region on the pJet1 vectorright after the linearization restriction site. Note that the constructsPL90, H8 and B9 are within the vector pJet1. We recovered anapproximately 400 base pair long fragment which we sequenced. Thesequence is shown in FIG. 8.

FIG. 8 shows an approximately 400 base pair fragment that indicates theexemplary linearized PL90 vector is stably integrated within the genomeof Nannochloropsis oceanica. We thus conclude that the exemplary vectorspresented herein successfully drive expression of genes inNannochloropsis oceanica.

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, and notlimitation. Thus, the breadth and scope of a preferred embodiment shouldnot be limited by any of the above-described exemplary embodiments.

What is claimed is:
 1. An expression vector for algal celltransformation comprising the nucleotide sequence of SEQ ID NO:2.
 2. Theexpression vector of claim 1, wherein a promoter in the expressionvector modulates the expression of a gene governed by the promoter inresponse to light intensity.
 3. The expression vector of claim 2,wherein the promoter in the expression vector increases the expressionof the gene governed by the promoter in response to a decrease in lightintensity.
 4. The expression vector of claim 2, wherein the promoter isa Vcp promoter.
 5. The expression vector of claim 1, wherein the algalcell is of algal genus Nannochloropsis.
 6. The expression vector ofclaim 1, wherein the expression vector is at least partially expressedas part of a transformed algal cell.
 7. An expression vector for algalcell transformation comprising the nucleotide sequence of SEQ ID NO:3.8. The expression vector of claim 7, wherein a promoter in theexpression vector modulates the expression of a gene governed by thepromoter in response to light intensity.
 9. The expression vector ofclaim 8, wherein the promoter in the expression vector increases theexpression of the gene governed by the promoter in response to adecrease in light intensity.
 10. The expression vector of claim 8,wherein the promoter is a Vcp promoter.
 11. The expression vector ofclaim 1, wherein the algal cell is of algal genus Nannochloropsis. 12.The expression vector of claim 1, wherein the expression vector is atleast partially expressed as part of a transformed algal cell.